Rat Data

Abstract

Introduction

Human DNA is damaged up to one hundred thousand times per day through exposure to DNA damaging agents such as reactive oxygen species, radiation, chemicals and endogenous metabolic by-products. Defective or inefficient repair of the most lethal kind of DNA damage, DNA double strand breaks which affect both strands of the DNA has been linked with early- onset cancer syndromes, advanced ageing and other neurological and immunological conditions. In Australia, current cancer risk screening is population-based for target groups at risk of breast, bowel and cervical cancer. For individuals identified as having an increased risk of a familial cancer syndrome such as Hereditary Breast and Ovarian Cancer Syndrome, genetic screening is available. However, population-based screens and genetic screening cannot predict the age of onset of disease or risk of early-onset disease. Furthermore, as only 5-10% of all diagnosed cancer cases are linked to hereditary cancer predisposition syndromes, there is a gap in cancer risk screening for individuals that do not have a hereditary cancer predisposition syndrome and do not fit the criteria for population-based screening. This PhD investigated the potential use of the DNA double strand break marker, -H2AX, for individualised assessment of DNA double strand break repair for its potential use in future clinical cancer risk screening.

Methods

This PhD thesis assessed DNA double strand break damage and repair in three experimental models. The first, a Wistar rat model which is examined in Chapter 4, investigated the link between DNA damage and repair within three haematopoietic tissues: bone marrow, peripheral blood mononuclear cells and spleen. This chapter also explored how ageing influences DNA damage and repair using sibling rats that had their samples analysed at ages 3 months and 15 months. This model also investigated the influence of biological sex on DNA damage and repair. The second study (Chapter 5) explored DNA damage and repair capacity collected from 45 healthy human participants ranging from ages 18-75. The third study (Chapter 6) was a pilot experiment that assessed how a previous history of cancer influences DNA damage and repair using the same series of techniques. The novel factor within this study is the inclusion of DNA double strand break assessment at three-time points; an initial baseline, after two-hours of DNA damaging agent (etoposide) treatment and following a one-hour post etoposide wash-out. All three models used flow cytometry assessment of staining of DNA double strand breaks with - H2AX antibody. Phosphorylation of the histone protein complex from H2AX to -H2AX was labelled with the fluorophore Alexa Fluor 488 and quantified via flowcytometry using gates that detected cells positive for -H2AX labelling. Quantification was calculated by the mean fluorescence intensity of -H2AX and percentage change in the mean fluorescence intensity of -H2AX.

Results

This research found that -H2AX has potential for use clinically for the assessment of DNA double strand break damage repair. This research also determined a standardised method for the assessment of DNA double strand break repair within bone marrow, peripheral blood mononuclear cell (PBMC)s and spleen and included verification of results in PBMC studies across specimens. Through optimisations and validation of methodology, this research found that for successful assessment of DNA double strand break repair assessment in primary cell culture, cell cultures must be treated with a proliferation stimulant for 72 hours prior to DNA damage induction with a chemotherapeutic DNA damaging agent. This research also found that the optimal time for DNA double strand break repair assessment is one-hour following a two-hour treatment with etoposide.

In the Wistar rat model, advanced age was linked to a significantly increased mean fluorescence intensity of -H2AX within baseline sampling of bone marrow and PBMCs. Significant retention of -H2AX was also seen following a one-hour fresh media incubation in bone marrow samples. Non-significant increases in -H2AX following a one-hour fresh media incubation were also observed with advanced age in PBMCs, which demonstrated translational potential for use within human studies. The human model of DNA double strand break damage and repair within PBMCs, found that males had a significantly higher mean fluorescence intensity of -H2AX following a two-hour incubation with etoposide when compared to females in the test cohort (p<0.05). The mean fluorescence intensity of -H2AX was also significantly increased in males compared to females after a one-hour incubation with fresh media in the test cohort (p<0.001). Males aged under 50 years had significantly higher mean fluorescence intensity of -H2AX than females aged under 50 years in the test cohort following removal of the DNA damaging agent etoposide and one hour incubation with fresh media in the test cohort of 16 participants (p<0.001) and the extended study (p<0.05). Increased mean fluorescence intensity of -H2AX following a one-hour fresh media incubation was also observed in males aged under 50 years compared to males aged over 50 years of age in the test study (p<0.05). No significant differences in mean fluorescence intensity of -H2AX were observed between females age under and over 50 years in the test or extended group.

A link between participants with a previously diagnosed early-onset cancer and ineffective DNA repair, termed a ‘negative DNA repair score’ DNA double strand break repair score was found in the pilot study investigating early-onset cancer and DNA double strand break repair. In this study, 21% of participants that were identified as having inefficient DNA repair had a history of early-onset cancer. In contrast, there were no participants with a history of early-onset cancer identified in the effective DNA repair group.

Conclusions

This study developed and optimised a standard method for assessing DNA double strand break repair within PBMCs in Chapter 3. This methodology also proved successful within bone marrow and spleen derived cells across species (Chapter 4 and 5). Within this standardised method, it was found that primary cell culture must be stimulated to proliferate with a final concentration of 5 µg/mL of phytohaemagglutinin before induction of DNA damage. This study also found the optimal DNA double strand break repair window to be one-hour post DNA damaging agent removal and wash-out. Through optimisations and development of a standard method for DNA double strand break repair assessment which proved effective across tissues and species, this study adds a novel contribution to the field of DNA repair. The outcomes detailed in Chapter 4 demonstrated in rats that PBMCs may be an effective surrogate tissue for DNA double strand break repair assessment in haematopoietic derived tissue. This suggests that the DNA double strand break response may be systemic and opens opportunity for exploration of the link between the DNA double strand break response within all tissues compared to PBMCs. Within Chapter 4, it was demonstrated that the DNA double strand break response is influenced by age. Basal DNA damage measured via mean fluorescence intensity of -H2AX was significantly increased in bone marrow and PBMC samples collected from 15-month-old rats compared to their 3-month-old siblings. In bone marrow samples collected from 15-month-olds rats had significantly increased mean fluorescence intensity of -H2AX after removal of the DNA damaging agent and a one-hour incubation with fresh media compared to 3-month-old rats (p<0.01). This was also observed in PBMC, although the trend was non-significant. Furthermore, the finding that PBMCs are an effective surrogate tissue that is directly affected by ageing, invited further investigation into the DNA double strand break repair response within human models explored in Chapters 5 and 6.

Findings from Chapter 5 demonstrated that in PBMC, males have a significantly increased mean fluorescence intensity of -H2AX compared to females in a controlled test group of 16 participants (p<0.05). Following a one-hour incubation with fresh media males also had significantly increased mean fluorescence intensity of -H2AX compared to females within the test group. In addition, young males (<50 years) had significantly higher mean fluorescence intensity of -H2AX compared to young females (<50 years) following a one-hour incubation with fresh media in the test group (p<0.001) and extended cohort. Increased mean fluorescence intensity of -H2AX was observed in males under 50 years of age following a one-hour fresh media incubation, compared to males over 50 years within the test study (p<0.05). These findings suggest that biological sex has an impact on the DNA double strand break repair response and warrants further investigation into the possible implications in cancer risk screening. A pilot study of the influence of a history of early-onset cancer was investigated in Chapter 6. Within this Chapter, a negative DNA repair score was associated with participants that had an early-onset cancer. Therefore, the findings of this study suggests that assessment of DNA double strand break repair capacity through -H2AX staining in response to laboratory induced DNA double strand breaks may be a potential tool for clinical cancer risk screening.

This dataset relates to the data collected from the rats used in this study.